We use an exocrine accessory reproductive gland of a male insect as a model for the study of cytodifferentiation. Our project concerns cell cycles, the commitment of cells toward new programs, and the expression of those programs. Some of the developmental transitions are dependent upon hormonal signals while others appear to be autonomous. The tubular accessory gland of male mealworm beetles first appears as a mesodermal rudiment in the last instar larva and becomes an elongate sac in the adult. During metamorphosis, the gland passes through phases of primary organogenesis in the prepupa, of cell proliferation in the pupa, of cell specialization in the late pupa and of terminal differentiation in the adult. The mature gland has a single type of secretory cell and exports a few classes of differentiation-specific soluble proteins which are enclosed in the spermatophore. We can score the extent of differentiation by morphology, electrophoresis, leucine incorporation, and immunochemistry. For mealworms, the titres of ecdysteroids have been measured during metamorphosis. There is a major peak in the mid-pupa which is correlated with G2 arrest and the expression of the adult cuticular program in the sternal epidermis. In contrast, when this ecdysteroid peak acts on the mesodermal tubular gland there is high mitotic activity and commitment toward the adult program. Expression of that program follows several days later. We can culture the gland in vitro or in vivo (transplants). The mid-pupal mitoses and the commitment will occur in vitro only when physiological levels of acdysteroid are present. In this proposal, we will first work out the kinetics of the cell cycle in vivo and then determine whether the ecdysteroid-treated cultures mimic those kinetics in vitro. Preliminary evidence indicates a long G2 during which ecdysterone acts to trigger mitoses. We will improve our immunochemical probes and then compare dose-levels, timing, and response of the ecdysteroid effects on cell cycles with that on commitment. Finally, we will ask whether the mitoses are prerequisite to reprogramming. Our model of cytodifferentiation is a model of insect reproductive function. Without efficient sperm transfer, most deleterious insect populations would not survive.